THE BREAKING-UP OF HIBERNATION IN THE 
CODLING MOTH LARVA 


BY 


MYRON THOMAS TOWNSEND 
B.S. Bates College, 1918 
M. S., University of Illinois, 1921 


A DIGEST OF A THESIS 
SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS 
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN ZOOLOGY 
IN THE GRADUATE SCHOOL OF THE UNIVERSITY 
OF ILLINOIS, 1925 


Reprinted from the Annals of The Entomological Society of America, 
Vol. XIX, No. 4. Pages 429-439 


THE BREAKING-UP OF HIBERNATION IN THE 
CODLING MOTH LARVA.* 


M. T. TowNnsEND 


INTRODUCTION. 


Hibernation has been frequently studied with a view to 
determining what factor or factors were responsible for pro- 
ducing the dormant condition. In the present paper the 
problem has been attacked with a view to determining what 
brings about the break-up of the dormancy in spring. The 
experimental data thus gained indicate that hibernation in 
the codling moth is more than a condition of passive resistance 
to cold weather and other unfavorable conditions. During the 
cold winter months changes are going on in the tissues of the 
animal which are necessary for the further functioning of the 
organism and hibernation is a necessary stage in the life history 
of the insect. 

The experimental data given below, when correlated with 
the work of others, justify the theory that the most important 
factor in the breaking up of hibernation in cold-blooded animals 
is the reabsorption of water by the tissues. This reabsorption 
speeds up the enzyme activity of the tissues and the animal 
becomes active again. A return of warm weather in the spring 
is of course also important, but this factor is always experienced 
by a hibernating animal as the season advances. Whether 
the animal can absorb the requisite amount of water depends 
upon the local situation in which it hibernated, and without 
this addition of water, the enzymatic baa ceares necessary for 
renewed activity cannot take place. 

The writer is indebted to Dr. V. E. Sheitord under whose 
direction the work was done and to other members of the 
Zoology Department at the University of Illinois for help and 
encouragement during its progress. 


* Contribution from the Zoological Laboratory of the University of Illinois 
No. 289. Submitted in the partial fulfillment of the requirements for the Degree 
of Doctor of Philosophy in Zoology in the Graduate School of the University of 
Illinois. 


429 


\ 54974 


430 Annals Entomological Society of America [Vol Xie 


MATERIALS AND METHODS. 


Codling moth larve of the second generation were collected 
in the vicinity of Urbana, Illinois, during the summer of 1923. 
Six-inch tar paper bands were tacked around the tree trunks 
for collecting the larve, and these were examined from time 
to time during the fall. The larve were removed from their 
cocoons with forceps and dropped into small cardboard boxes, 
which had been previously prepared by placing in the bottom 
of each a layer of small glass tubes. The tubes were approxi- 
mately 14 inch in diameter and one inch long, and were stood 
on end and packed tightly into the bottom of the box. The 
larve showed a strong tendency to crawl into the tubes and 
spin cocoons there, and these tubes of cocoons were later re- 
moved from the boxes for use in the experiments. 

A field note-book was kept during the season, containing 
data as to when bands were placed on the trees, when larve 
were collected, number of rainy days, cold nights, etc. 

Experiments were performed in the vivarium building, of 
the University of Illinois, using three temperature chambers, 
one running at 0°C., one at 10°C., and one at 30°C. Recording 
thermographs were operated constantly in each chamber and 
the records showed no variations which might have affected 
the work. It was found that storage at 0°C. caused the lowest 
mortality among the larve, altho as shown later in the paper, 
exposure to 10°C. for a time was most conducive to pupation 
and later progress. 

Records of pupations, emergences, etc., were kept for in- 
dividual larve by labeling the tubes either with a glass pencil 
or a small sticker. 


DISCUSSION. 


It has been shown by numerous workers that the beginning 
of hibernation in cold-blooded animals is often marked by a 
reduction of the water content of the organism, and it is equally 
true that the breaking-up of hibernation in these forms is 
usually marked by a taking in of water. This difference in 
water content is perhaps the most universally recognized 
difference between the hibernating and non-hibernating animals 
among the insects and related groups, and may well serve as 
the starting point for a theory of hibernation. 


1926] Townsend: Hibernation in Codling Moth Larve 431 


Tower (1906) described the preparation for hibernation 
in the potato beetle and showed that the animal eliminated 
27% of its weight as watery material during a period of three 
to ten days and then burrowed in the ground and hibernated. 
In the spring this water was regained and the protoplasm 
became more liquid again. Breitenbecker (1918) showed 
that dessication of potato beetles caused them to pass into a 
state of “induced” hibernation. According to Bodine, (1923) 
in grasshoppers which hibernate, such a condition was marked 
by a lowering of the water content to a minimum, and the 
breaking up of hibernation was marked by a return to the nor- 
mal condition in this respect. 

Several workers have shown that the addition of water 
tends to break up dormancy. Thus Breitenbecker found that 
the addition of water to the soil would cause hibernating potato 
beetles to emerge, if the temperature was above 14°C. If the 
soil was never wet the beetles died instead of emerging. Along 
this same line Weese has shown that young spiders emerge 
from the winter cocoons only under conditions of high humidity 
of the surrounding air. 

In investigating the winter brood of the codling moth 
larve in the vicinity of Urbana, Illinois, I found that soaking 
the larve at intervals (2 hours per week), caused them to 
pupate more readily than if they were not soaked, Pupation 
in this case marks the end of the dormant period and the re- 
turn to functional activity. The main points of this phase 
of the work are shown in Table 1 from which the following 
conclusions may be drawn: 


1. Soakings increase the per cent of pupation, whether the 
water is applied at low temperature (10°C.) or at higher tempera- 
ture (30° C.) or both. 


2. Soakings at 10°C. shortens the length of time to pupation. 


3. Soakings at 30°C. shortens the length of time from pupa- 
tion to emergence. 


From these conclusions it seems evident that some process 
goes on at 10°C., is affected by soakings and is intimately 
concerned with pupation. Also some similar process goes 
on at 30°C., is affected by soakings and is intimately concerned 
with emergence or in other words with the later stages of meta- 
morphosis. Such soakings would be normally experienced 


432 Annals Entomological Society of America [Vol. XIX, 


by the animals during the spring rains, and probably are effec- 
tive by reason of their action upon the enzymes of the body 
tissues and fluids. 

That water-content has a marked effect upon pupation 
processes is further shown by Table 2 where the data on all 
larvee experimented upon have been tabulated with regard 
to this one factor, disregarding other points in the histories 
of the various groups. Reference to column 3 shows that the 
greatest percent of pupation occurs among animals soaked 
frequently at the rate of two hours per week. 


TABLE I. 


Showing effects of soaking hibernating codlin moth larve at different tempera- 
tures. Storage temperature 10°C. Pupation temperature, 30°C. 


1 2 | 3 a 5 6 a 8 9 


Num- 
2-hr. Num- Days ber Days | Number} Days | Number 
soak- ber % to pupae to emer- to of dead 
ings 2-hr. | Pupa- | pupa- on emer- gences | death of] larvae 
during | soak- ted | tion at| which | gence |on which} larvae jon which 
storage | ings at 80°C. | column] at 30°C. |column6 | at 30°C. | column8 
307: 4 is is based is based 
based 
5 5 36.0 23.0 8 8.0 Ys 30.6 14 
0 5 33.0 26.0 16 7.0 9 14.8 10 
5 0 32.7 Lom 17 10.8 14 | 25.8 11. 
0 0 6.6 29.0 1 9.0 1 21.5 14 


The effect of water-content upon enzymatic action is well 
known. According to Fischer, (1907), ‘‘In a concentrated 
solution the point at which the reaction (of digestive enzymes) 
comes to a standstill is reached sooner than in a more dilute 
one. In fact most fermentation mixtures which have come 
to a standstill will go further only if water is added.” 

Bayliss in his monograph on enzyme action emphasizes 
the importance of water in such processes. According to this 
author, ‘‘The greater number of enzymes have a hydrolytic 
action, and their activities are as a rule manifested in the 
presence of water.’’ Again “‘an enzyme action comes to an 
end or equilibrium point owing to the accumulation of the 
products of the reaction, so that by dilution or removal of the 
products the reaction may be caused to go on further.” 


1926] Townsend: Hibernation in Codling Moth Larve 433 


The breaking-up of dormancy in the codling moth larva 
is marked by the metamorphosis of the insect. Such meta- 
morphosis consists morphologically of two processes, namely 
a process of autolysis of larval tissues, followed by a process 
of growth and development of adult organs. From a chemical 
standpoint, then, pupation of the codling moth larva in the 
spring is initiated by a process of autolysis. This autolysis 
is probably due to the action of enzymes, an hypothesis to be 
tested more carefully by later work. A consideration of some 
data available at this time lends indirect support to this view. 


TABLE II. 


Summary of results of all codling moth experiments grouped together to show 
effects of moisture alone, pupation experiments at 30°C. 


1 2 3 4 5 6 7 8 9 
Days | Num- | Days | Num- | Days | Num- 
Storage No. a to ber to ber to ber of 
Conditions | larvae | pupa- | pupa- | pupae | emer- | emer- | death, | larvae 
ted tion | col. 4 | gence | gences | larvae on 
col col. 8 
Nevér soaked 76 24.6 19.9 15 10.6 8 30 45 
Soaked once 
for 15 hrs.. | 1148 8.5 22.4 48 11.3 31 32.9 274 
Soaked sev- 
eral times 
at rate of 2 
hr.perweek.| 350 50.8 20.7 123 9.5 90 26.2 171 


Recent writers have emphasized chiefly the action of enzymes 
or of acids in bringing about the breaking down of such tissues. 
Thus Weinland (1909) found that the formation and destruc- 
tion of fat in the larva of Calliphora was a process governed 
by an equilibrium condition under the action of enzymes. 
Simon (1904), Cole (1919) and others mention enzymes as being 
responsible for autolytic processes in tissues. On the other 
hand Bishop (1923), has shown that the accumulation of acid 
in the body of the honeybee larva was probably the primary 
cause of autolytic processes there, and that enzymes were not 
important until later. 

Whether autolysis in an insect larva is brought about by 
acids or by an enzyme, the addition of water would be expected 


434 Annals Entomological Society of America [Vol. XIX; 


to speed up the action and increase it. Thus, Bishop inghie 
discussion of the metamorphosis of the honey-bee larva, where 
autolysis is due largely to acid, says ‘‘when the tissues are 
diluted with pure water, the buffering action of the mixture 
is lowered, and a slight excess of acid in one tissue or another 
would accelerate its rate of autolysis.” 

We have already noted that dilution has a marked effect 
upon enzyme action. It is well to remember in this connection 
that the codling moth larva and the bee larva pupate under 
quite different conditions of temperature, etc. 


TAB HL ie 


Showing rate of pupation in samples of codling moth larve from the same collection, 
placed under pupation conditions on successive dates during the winter. The 
first column shows storage temperature and days in storage. At the close of 
storage the larve were placed in 30°C. for pupation. The number of larve 
used was 10 except in one case where 18 were used. The stock of the second 
item marked with an asterisk, 29.38% pupated at 22°C., indicating that the 
higher temperature (380°C.) is not essential. However, all this group had 
undergone preparation for pupation at the lower temperatures experienced 
in the field before collection. 


Te da. % pupation Da. to pupation | Da. pupal Life 

22 0 Bt ener ee 30 22 12 
Fe Oe co atts shee toe 20° A ee re 

22° SI OR eintetexe eects eio% Qa SAE i ete oeaa 4 ah re 

10% Oot cea ar 50 23 Ait 

10° 58.vodacgorenne 30 21 9 

10°; 100 ic. aa 27.7 142s es a 

"Oe hd eoe nets 40.0 21 9 

Ore OS es aoa Wetec part 20.0 19 11 

O%s LOO: Bee peel eee 25.0 [4 et" \o ee 


That the autolytic process in the hibernating codling moth 
larva is probably due to an enzyme rather than to accumulation 
of acid seems probable from some of my experimental data, 
altho as yet no real chemical tests have been made. Experi- 
ments show that larve kept at low temperatures for consider- 
able periods do not pupate but do undergo some sort of prepara- 
tion for pupation, so that when placed at 30°C. later, they 
pupate more rapidly as a result of their treatment at low tem- 
perature. Thus, as shown in Table 8, larve collected and stored 
at 10°C. or O°C. showed increased speed of pupation when 
placed at 30°C, later in the season. Speed of pupation. is 
indicated in column 3 of Table 3. Such exposure to low tem- 


2 


1926] Townsend: Hibernation in Codling Moth Larve 435 


peratures appears to be the usual pre-requisite to the breaking 
up of hibernation in the codling moth larva, and evidently 
allows some sort of preparation process to go on. 

It will be noted from Table 3, column 2, that larve stored 
at 22°C over a period of time lost their power to pupate. This 
suggests that the necessary preparation cannot go on at this 
temperature. Some data on this point are available. Forty 
larve collected August 11 were never exposed to temperatures 
below 13°C. either in the orchard or the laboratory. Later 
they were placed at 30°C. but showed no pupation. 

195 larve which had never been exposed to temperatures 
below 22°C. in the laboratory showed only 314% pupation 

TABLE IV. 


Showing effects of exposure of codling moth larve to low temperatures for varying 
periods of time. Pupation temperature, 30°C. 


ia lb P t of 
Number Larvae Low Temperature Tenipers uke Panaon 
106 Oz: 7-14 days 6.6 
74 (OLE: 30 days 28.0 
220 10°C. 7-14 days 25.4 
‘de ites 30 days 8.3 


when placed at 30°C., and this slight percent may be explain- 
able by a slight exposure to low temperature in the orchard 
before collection as shown by field records. 

Evidently then in the hibernating larva, some process 
which is a prerequisite to pupation goes on only at low tem- 
peratures. Table 4 gives some data regarding the effect of 
low temperatures on this process. It shows that the process 
goes on rather slowly at 0°C. and considerably faster at 10°C. 
indicating a maximum activity for the process at about 10°C. 
Furthermore exposure to 10°C. if continued long enough may 
prove detrimental. Exposure for as long as 30 days may 
undo some of the work accomplished during the first two 
weeks. 

The fact that preparation for pupation does go on slowly 
at as low a temperature as O°C. is of considerable interest, 


436 Annals Entomological Society of America [Vol. XIX, 


and indicates that the metabolic activity of the animal is not 
readily brought to a standstill by winter conditions. The 
effects of exposure to different temperatures is further shown 
in Table 5, where the data regarding all larve used in the ex- 
periments have been tabulated with regard to this one factor. 
As shown in Column 2 of this table, percent of pupation was 
highest in larve stored at 10°C., lowest among those at 22°C, 
It seems likely that if the process of autolysis of larval 
tissues was due to acidity it would be speeded up by higher 
temperatures, whereas the data given above indicate that in 
the winter brood of codling moth larve the process does not 


TABLE V. 


Summary of results of all codling moth experiments grouped together with regard 
to storage temperature alone. Pupation and larval death temperatures, 30°C. 


1 2 3 4 5 6 Uf 8 9 10 
Da Days Aver- 
Sree % y No. Pupal No to Dead Total % of 
age | Pupa- upa- | Pupae Life, ein death | larvae larvae. | oe 
8 tion fe col. 3 | Days 5 of col. 3 tality, 
larvae storage 
D226" 8.1 21.9 40 8.4 28 35.6 358 1141 56 
102C@25), 252.6 Diva 146 9.5 108 26.9 233 452 38 
OLC a eeote? 17.8 10 9.6 6 28.1 16 32). 0 


go on at all at temperatures above 22°C. An enzyme on the 
other hand might well have a maximum activity at a tempera- 
ture as low as 10°C. especially in a cold-blooded aimal like 
an insect. Accordingly we may suppose that in the hiber- 
nating codling moth larva autolysis of larval tissues is brought 
about by an enzyme which works best at temperatures around 
10°C. Such a hypothesis can be tested by later work. On 
this basis the exposure of the animal to low temperatures 
during the hibernating period is not a case of mere resistance 
to unfavorable conditions but in part at least is a period neces- 
sary for certain vital functions to go on. 

If the above hypothesis is correct, i. e., that changes in larval 
tissues are the result of an enzymatic process, affected by 
soakings and having a temperature maximum of about 10°C., 
we should expect that a combination of these two factors 


1926] Townsend: Hibernation in Codling Moth Larve 437 


in the environment would give the maximum effects. Table 6 
shows the available data on this point.. The table is incomplete 
as regards some groups, but the following conclusions may 
safely be drawn from it. 


1.—Percent of pupation at 30°C. is— 
Greatest after exposure to 10°C., soaked frequently 


Next(lower) “ SNe a0 Cas ab Once. 
Next(lower) “ E22 (Or $ be 
Least « «  & 99°C., dry. 


2.—Number of days to pupation at 30°C. is— 

Shortest after exposure to 10°C., soaked frequently. 

Next - sibs tan’ vag toe Sie OUCE, 

Longest “ “ “ LO2GE, “ “ 
3.—Number of days from pupation to emergence at 30°C. is— 

Shortest after exposure to 22°C., soaked once. 

Next “ “ “ 0° - “ “ 

Longest . et BU hl Oe “ frequently. 


TABLE VI. 


Showing effects of storage temperature and moisture upon hibernating Codling 
moth larve. All pupation experiments at 30°C. 


| 2 3 4 5 6 ( 8 9 10 
No. 
i, Conditions No. % Days No. D oe emer- ae No. 
Temperature and * |pupa- | pupa- pupae gen- larvae 
: larvae pupa- emer death 
moisture) ted | ted ae Lab eoome eS liar eae col. 9 
cole? 
AS PIES Ss ces tine, 38 12 | 34.2; 19.3] 12 | 10.4 ~ | 30.5 filg 
10°C. Soaked once , 
for 15 hrs.....| 58 8 | 138.8 | 25.0 8 9.0 7 | 24.1] 38 


10°C. Soaked sev- 
eral times 2 
hrs. per week.|} 350 | 178 | 50.8 | 20.7 | 123 9.5} 90 | 26.2 | 171 


22°C. Soaked once 
fore anita a. 1,090 | 90 | 12.1] 21.9] 40 SoG) 24 lod) 230. 


We see, then, that the greatest percent of pupation and 
fastest speed in the process goes on at 10°C. soaked frequently, 
and this is in agreement with our hypothesis. In other words 
frequent small soakings at 10°C. furnish optimum conditions 
for the enzymatic processes which precede pupation. The 
cold, soaking rains of winter and spring would furnish ideal 
conditions for preparing the animal for renewed activity as 
soon as the weather warms up a little. 


438 Annals Entomological Society of America [Vol. eis 


The summer brood of codling moth larve do, however, 
pupate at higher temperatures, and such an enzyme would not 
be able to work under such conditions. This is obviously a 
different case, since continued exposure to summer tempera- 
tures has no effect upon the winter brood, as far as pupation 
is concerned. We may perhaps explain the autolysis of larval 
tissues in this case on the basis of acidity. We have already 
noted that Bishop, (1923), found that in the bee larva autolysis 
is due largely to the increased acidity of the tissues due to the 
activity of spinning, assimilation of fats, etc. The case of the 
bee larva is similar to that of the summer brood of the codling 
moth larve in that the metamorphosis is rather rapid and 
goes on at a rather high temperature. The case of the summer 
brood has not yet been exhaustively studied from this point 
of view. 

_In the case of the codling moth and doubtless in others 
not yet studied in this way, it is evident that addition of water 
to the tissues tends to break up the dormant period and renew 
life processes. The renewed activity of the animal may be 
shown in various ways in different species—by metamorphosis 
in the case of the codling moth; by growth and activity in the 
case of young grasshoppers or in certain spiders; or in the case 


of animals which hibernate as adults, by simply renewed - 


activity, especially as regards the sexual organs. Perhaps 
further work will show that in these latter cases addition of 
water has a marked effect upon the enzymatic processes in 
the organs of generation which are usually most active in the 
spring. 


SUMMARY. 


1.—The break-up of hibernation in the codling moth larva 
is hastened and aided by the addition of water to the tissues, 
such as normally takes place during rains. This water, by 
diluting the fluids, probably speeds up the enzyme action in 
the animal’s body, and the result is a renewal of metabolic 
processes, pupation, and general activity. The frequency 
of soaking is of much importance. 

2.—During the exposure to rather low temperatures in 
winter and spring the tissues of the winter brood of codling 
moth larve undergo a preparation for pupation which marks 
the renewal of activity in spring. 


otty 


1926] Townsend: Hibernation in Codling Moth Larve 439 


3.—The preparation process does not go on at temperatures 
aeeidaen as 22 CG. 910°C. is more favorable for it than 0°C. 
It may be explained by assuming the presence of an autolytic 
enzyme in the tissues, which works best at a temperature 
near 10°C. 


LITERATURE CITED. 


Bayliss, W. M. The Nature of Enzyme Action. p. 12, 1911. 

Bishop, G. H. Journal of Biological Chemistry, LVIII, No. 2, 1923. 

Bodine, J.H. Jour. Exp. Zool. XXXII, 1921; XX XVII, 1923. 

Breitenbecher, J. K. Yearb. Carn, Inst. Wash, No. 10, 1911; Carn. Inst. Pub. 
No. 263, Appendix, 1918. 

Cole, S. W. Practical Physiological Chemistry. 1919. 

Simon, C. E. Textbook pf Physiological Chemistry, 1904. 

Tower, W.L. Carn. Inst. Publ. No. 48, 1906. 

Weese, A.O. Animal Ecology of an Illinois Elm-maple forest. Ill. Biological 
Monographs IX No. 4. 1924. 

Weinland, E. Zeitschrift. f. w. Zool. XIV, 1864. 


V ita 
MYRON THOMAS TOWNSEND 


Born May 8, 1897, West Hampton, Massachusetts. 
B. S. Bates College, Lewiston, Maine, 1918. 
Student, Massachusetts Institute of Technology, Cambridge, Massachusetts, Sum- 
mer, 1918. 
Graduate Student, University of Illinois, 1919-1924. 
Graduate Student, Puget Sound Biological Station, Summer of 1920. 
M. S. University of Illinois, 1921. 
Graduate Student, University of Chicago, Summer 1923. 
Part time assistant in Zoology, University of Illinois, 1919-1923. 
Instructor in Zoology, St. Procopius College, Lisle, Illinois, Summer 1921. 
Fellow in Zoology, University of Illinois, 1923-1924. 
Member of--- 
American Association for the Advancement of Science. 
Ecological Society of America. 
Entomological Society of America. 
Sigma XI. 


Illinois State Academy of Science. 


3 0112 072841627 


UTAH r 


